Bacillus subtilis engineering bacterium for expressing MccJ25 as well as construction method and application of bacillus subtilis engineering bacterium

A technology of Bacillus subtilis and engineering bacteria, which is applied in the fields of genetic engineering, protein expression and functional verification, can solve problems such as the difficulty of avoiding endotoxins in Enterobacteriaceae, achieve intestinal colonization of live bacteria, increase application value, and avoid endotoxins the effect of the influence

Active Publication Date: 2022-06-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most of the secreted expression and actual production of MccJ25 use Gram-negative bacteria such as Enterobacter as the expression strain, and Enterobacter cannot avoid the influence of endotoxin
For Gram-positive bacteria, due to the restriction of the expression system of the bacteria itself, very few people use Gram-positive bacteria to express MccJ25

Method used

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  • Bacillus subtilis engineering bacterium for expressing MccJ25 as well as construction method and application of bacillus subtilis engineering bacterium
  • Bacillus subtilis engineering bacterium for expressing MccJ25 as well as construction method and application of bacillus subtilis engineering bacterium
  • Bacillus subtilis engineering bacterium for expressing MccJ25 as well as construction method and application of bacillus subtilis engineering bacterium

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Experimental program
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Effect test

Embodiment 1

[0035] 1. Experimental materials

[0036] (1) Biological materials:

[0037] Bacillus subtilis BS168 strain and WB800N strain (both purchased from Beina Biotechnology Co., Ltd.), Salmonella Enteritidis ATCC 14028 (Shanghai Beinuo Biotechnology Co., Ltd.) and PHT43 plasmid (Wuhan Miaoling Biotechnology Co., Ltd.), The primer synthesis and gene sequencing were entrusted to Guangzhou Aike Biotechnology Co., Ltd.

[0038] (2) Experimental reagents and culture medium:

[0039] DL-Threonine, Glycine, Tryptophan, Tween 80, Sorbitol, Mannitol, Trehalose;

[0040] 2×YT liquid medium: tryptone 16g / L, yeast extract 10g / L, sodium chloride 5g / L, sterilized at 121°C for 20min;

[0041] LB solid medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 5g / L, agar powder 15g / L, sterilized at 121°C for 20min;

[0042] Electroporation buffer: 0.5M trehalose; 0.5M sorbitol; 0.5M mannitol; 0.5mM MgCl 2 ;0.5mMK 2 HPO 4 ; 0.5mM KH 2 PO 4 .

[0043]Plasmid Extraction Kit (Guangzhou Meiji...

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Abstract

The invention discloses a bacillus subtilis engineering bacterium for expressing MccJ25 as well as a construction method and application of the bacillus subtilis engineering bacterium. The engineering bacterium is constructed by cloning a mcjABCD sequence which is modified by lasso peptide MccJ25 to bacillus subtilis. Wherein the sequence of the mcjABCD is as shown in SEQ ID NO. 1. The constructed bacillus subtilis engineering bacteria can successfully express the MccJ25, and the bacillus subtilis belongs to food-grade probiotics and can avoid the influence of endotoxin, so that the bacillus subtilis engineering bacteria can be used as probiotics for producing the MccJ25; meanwhile, the engineering bacterium also has a relatively strong inhibition effect on salmonella enteritidis and the like, and can be used in the aspect of antibacterial products so as to improve the application value of MccJ25.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, protein expression and function verification, and particularly relates to a Bacillus subtilis engineering bacterium expressing MccJ25 and a construction method and application thereof. Background technique [0002] MccJ25 is a bacteriocin class I antibacterial small peptide found in Escherichia coli. Its size is composed of 21 amino acids. Its lariat structure is a β hairpin structure formed by the 9th to 21st amino acids of the N-terminal. It runs through the 1st to 8th amino acids. It is formed by a cyclic structure formed by a single amino acid and maintains stability through covalent action. Therefore, mature MccJ25 has a certain tolerance to extreme environments. Usually, MccJ25 inhibits bacterial transcription by blocking the transport of bacterial RNA polymerase, or by participating in the inhibition of bacterial membrane respiratory chain, thereby killing sensitive bacteria or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N15/31C12P21/02C07K14/245A61K35/742A61K38/16A61P31/04C12R1/125
CPCC07K14/245C12N15/75A61K35/742A61K38/164A61P31/04Y02A50/30
Inventor 张广文冯赛祥廖明林敏谢倩梅江金飞代绘琳陈爱华
Owner SOUTH CHINA AGRI UNIV
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